The integrity of the vascular endothelium of the blood-brain barrier (BBB) is central to cerebrovascular homeostasis. Given the function of the BBB as a physical and metabolic barrier that buffers the systemic environment, oxidative damage to the endothelial monolayer will have significant deleterious impact on the metabolic, immunological, and neurological functions of the brain. Glutathione (GSH) is a ubiquitous major thiol within mammalian cells that plays important roles in antioxidant defense, oxidation-reduction reactions in metabolic pathways, and redox signaling. The existence of distinct GSH pools within the subcellular organelles supports an elegant mode for independent redox regulation of metabolic processes, including those that control cell fate. GSH-dependent homeostatic control of neurovascular function is relatively unexplored. Significantly, GSH regulation of two aspects of endothelial function is paramount to barrier preservation, namely, GSH protection against oxidative endothelial cell injury and GSH control of postdamage cell proliferation in endothelial repair and/or wound healing. This paper highlights our current insights and hypotheses into the role of GSH in cerebral microvascular biology and pathobiology with special focus on endothelial GSH and vascular integrity, oxidative disruption of endothelial barrier function, GSH regulation of endothelial cell proliferation, and the pathological implications of GSH disruption in oxidative stress-associated neurovascular disorders, such as diabetes and stroke.
Central to neurovascular homeostasis is the function of the blood-brain barrier (BBB). The BBB is a highly regulated interface between the systemic circulation and brain parenchyma and is comprised of a monolayer of brain capillary endothelial cells on the blood side and perivascular cells on the brain side of microvessels. The BBB functions to protect the parenchymal cells from fluctuations in plasma composition, such as during exercise and following meals, and against circulating neurotransmitters or xenobiotics capable of disrupting neural function [
On the luminal and abluminal membranes, specific transport systems regulate the transcellular traffic of small hydrophilic molecules, such as GLUT-1 and L-system carrier 1 in the transport of glucose or leucine, respectively, thereby providing a selective “transport barrier” that facilitates nutrient entry [
The glutathione/glutathione disulfide (GSH/GSSG) couple is the most abundant thiol redox system that plays a key role in the maintenance of the redox environment in cells [
The versatility of GSH in contributing to a myriad of cellular functions is notable in its role in detoxication reactions (e.g., hydroperoxide and xenobiotic catabolism), regulation of amino acid transport into cells, maintenance of native three-dimensional protein structure in biosynthetic/metabolic processes (e.g., prostaglandins D2 and E2 synthesis), serving as a cofactor for enzyme systems (e.g., glyoxalase I), and redox signaling. Thiol-disulfide exchanges and protein S-glutathiolation are mechanisms by which GSH modulates the oxidative modification of redox active cysteines within proteins and thereby regulates the activity of a variety of enzyme functions, including those controlling proliferation, differentiation, or apoptosis [
Intracellular GSH is differentially distributed among the various subcellular compartments of cytosol, mitochondria, endoplasmic reticulum, and nucleus wherein distinct redox pools are formed [
While the biological importance of metabolically unique GSH compartments in redox regulation of various endothelial cell functions [
One of the undesired consequences for an organism living in an aerobic environment is an increased potential for oxidative damage by reactive oxygen species (ROS). However, the ability to thrive within such an aerobic environment also implies an evolved capability to handle ROS-mediated tissue damage [
The recognition that ROS can serve as important mediators of cell signaling and that signal transduction may be mediated by ROS-induced GSH redox imbalance is major conceptual breakthrough in our understanding of GSH-dependent redox signaling [
GSH exerts profound effects on vascular endothelial function, which include endothelial barrier permeability [
Mechanisms of MG-mediated endothelial barrier dysfunction and its protection by GSH
A large body of evidence supports a role for S-glutathiolation in redox regulation of vascular function, ranging from cell signaling, apoptosis, protein folding, to cytoskeletal reorganization. In hypertensive vessels, the thiolation of endothelial nitric oxide synthase (eNOS) is pivotal in the redox control of vascular tone. The bioactive nitric oxide (NO) molecule plays a crucial role in normal endothelial function, including modulation of vascular dilator tone, inhibition of platelet activation, inhibition of leukocyte adhesion and migration, and inhibition of smooth muscle cell migration and proliferation [
Current evidence implicates the involvement of S-glutathiolation/deglutathiolation in apoptotic signaling. In TNF-
GCL-catalyzed
The promoters of GCLc and GCLm subunits share common elements and coordinate transactivation results in overall increase in subunit expression. Key mediators of GCL expression are the redox sensitive transcription factors, nuclear factor kappa B (NF-
Constitutive or induced posttranslational phosphorylation of GCLc further contributes to GCL control. In contrast to insulin and hydrocortisone, which induced GCLc gene expression [
The transcriptional regulation of GCLm is poorly understood. Current evidence shows that the human GCLm promoter also contained an ARE site that mediated Nrf2-dependent GCLm upregulation induced by
It is abundantly clear that oxidative stress induced by ROS such as
Control of paracellular permeability in the endothelium is a function of the intercellular endothelial adherens junctions (AJ) and tight junctions (TJ), a complex structure comprised of specific junctional proteins. The cadherins,
Carbonyl stress is the result of enhanced reactive carbonyl species (RCS) production and decreased carbonyl-scavenging capability, leading to tissue accumulation of reactive dicarbonyl species, such as methylglyoxal (MG). MG is produced from cellular glycolytic intermediates and can induce carbonyl stress through irreversible reaction with free arginine residues of proteins to form advanced MG-glycated end product (AGE) [
The crosslinking of MG and amino acids was shown to yield the
Altered cell morphology, aberrant cytoskeletal rearrangement, and ZO-1 loss were notable biological consequences of glyoxal, another sugar-derived aldehyde product. Additionally, glyoxal also elicited mitochondrial dysfunction, inhibition of DNA and cell replication, and cell cytotoxicity through protein carbonyl formation [
Cell cycle control is crucial for proper postdamage endothelial repair and growth. The mammalian cell cycle is characterized by a quiescent G0 phase of nondividing cells followed by cell entry into the cell cycle at G1 and progression through the S, G2, and M phases in response to environmental or cellular cues that overcome the biological constraint of a mitotic block [
Nuclear glutathione cycle and associated redox changes during cell cycle progression
The progression of cells through the cell cycle has been linked to dynamic changes in the intracellular redox environment particularly that of the GSH/GSSG redox couple from a more oxidized state prior to cell cycle initiation to a more reduced state throughout cell cycle until cell cycle exit after prometaphase and cytokinesis (Figure
A role for ROS in mitogenic signaling is underscored by the finding that treatment of serum-starved cells with the thiol antioxidant, N-acetylcysteine (NAC), elicited cell cycle arrest at G1, a delay of G0 to G1 progression that correlated with defective redox control [
Intracellular redox homeostasis is maintained by the thiol/disulfide redox systems of GSH/GSSG, thioredoxin (Trx/TrSS), and cysteine (Cys/CySS). The product of reducing potential and reducing capacity of the redox couples determined the cellular redox environment, which in most cells are largely governed by that of the GSH/GSSG couple [
Recent evidence suggests that the dynamic cytosol-to-nuclear GSH distribution was a crucial factor in cell cycle progression in that nuclear GSH accumulation provided an intranuclear redox environment that enabled proper regulation of redox signaling events during the various stages of the cell cycle [
As an organ that is highly dependent on oxidative metabolism for its energy needs, the brain is susceptible to tissue GSH imbalance and oxidative damage mediated by increased formation of free radical species and lipid peroxidation [
The findings that GSH levels in endothelial cells in culture increased during the lag phase, were elevated during the initial exponential growth phase, and then fell as cells become confluent [
Increased BBB permeability has been demonstrated in patients with type II diabetes [
It is well known that diabetes is associated with hyperglycemia, elevated oxidative and carbonyl stress, and low tissue and plasma levels of GSH [
Stroke is a cerebrovascular disorder wherein a blood clot or interrupted blood flow to a region of the brain leads to a rapid loss of brain function. Significantly, a lack or delayed flux of oxygen and glucose to the brain will result in neuronal death and brain damage. Clinical studies have shown that subjects at risk for stroke exhibited low tissue GSH levels and decreased GSH-to-GSSG ratio and that the restoration of normal cerebral GSH balance could be as long as 72 h after the ischemic insult [
The restoration of endothelial integrity after thrombotic or hemorrhagic stroke is crucial to preserving BBB function and neurovascular homeostasis. The proliferation of endothelial cells adjacent to the lesion or injury site is a pivotal step. Given the role of GSH in cell proliferation (Section
Reportedly, GSNO functions in cellular signaling [
The integral function of the microvascular endothelium underpins cerebrovascular homeostasis. ROS- and/or RCS-induced endothelial dysregulation is an underlying concern in barrier failure, and, as such, much research has focused on the use of antioxidants as a strategy to attenuate oxidative or carbonyl stress and restore monolayer function. The finding that GSH, a major cellular antioxidant, is able to afford cytoprotection supports the notion that antioxidant therapy is important in endothelial barrier preservation. In past years, more recent conceptual advances in redox cell biology have uncovered a fundamental role of GSH in signal transduction and redox signaling in cellular functions. Moreover, the finding that distinct pools of GSH exist in subcellular organelles that allow for independent redox regulation has revolutionized our thinking of GSH-dependent redox mechanisms in controlling metabolic processes. One such biological process is that of cell proliferation. In the context of enhanced endothelial proliferation and self-repair surrounding lesion sites in response to systemic cues, for example, growth factors, little is known of a role for GSH. The dynamics of cytosol-to-nuclear GSH distribution appears to be pivotal in governing cell cycle responses. The notion that cell proliferation and growth can be a relevant biological process for monolayer repair/restitution following endothelial injury in much the same way as epithelial cell restitution/proliferation restores postinjured epithelium suggests exciting new avenues for future research in endothelial biology. Importantly, an understanding of GSH control of endothelial cell proliferative potential under different oxidizing conditions and plasma GSH levels will expand our perspective for future development of therapeutic strategies. Targeting endothelial restoration after oxidative insult and tissue damage is likely to be clinically relevant to the neurovascular disorders of diabetes and stroke and additionally could have broader implications for neurodegenerative and neurological disorders as well.
advanced glycated end product
Adherens junctions
Activator protein-1
Activator protein-2
Antioxidant response element
Blood-brain barrier
Cyclin-dependent kinases
Central nervous system
Cysteine
Cystine
Redox potential
Endothelial nitric oxide synthase
Electrophile responsive element
GCL catalytic subunit
GCL modifier subunit
Glucose-related protein
Glutaredoxin
Glutathione
S-nitrosoglutathione
S-nitrosoglutathione reductase
Glutathione disulfide
Hydrogen peroxide
Heat shock protein 27
Heat shock protein 60
Inositol-1,4,5-trisphosphate
IP3 receptor
Methylglyoxal
N-acetylcysteine
Nuclear factor kappa B
Nitric oxide
NO synthase
Nuclear factor E2-related factor 2
Superoxide anion radical
Reactive carbonyl species
Reactive oxygen species
Streptozotocin
Tight junctions
Thioredoxin
Zonula occluding protein
W. Li and C. Busu, contributed equally.
The authors declare no conflict of interests.
Research in the authors’ laboratory is supported by a Grant from the National Institutes of Health, DK44510.